How a neural circuit encodes, transmits, and processes information to make behavioral decisions is far from completely understood. Knowledge of the neurotransmitter usage, and thus the potential functional capacity, of each neuron in a neural circuit will significantly facilitate neural circuit analysis, as will enhancingthe spatial precision with which neuronal subsets can be functionally manipulated. This proposal aims to develop a set of neurotransmitter-specific fly strains for each Drosophila neurotransmitter that utilizes an intersectional genetic method to enable: 1) the complete definition of the neurotransmitter usage of all Drosophila neurons; and 2) neurotransmitter-specific silencing and transgene targeting to intersecting neuronal subsets within a GAL4 expression pattern. The method involves inserting B3 recombinase target sites (B3RTs) into neurotransmitter- specific genes such that the function of the gene is maintained and essential coding sequences are flanked. Subsequent expression of the B3 recombinase using a UAS-B3 recombinase transgene controlled by a GAL4 driver results in excision of the DNA between the B3RTs within all neurons in which the GAL4 driver expresses. The excision creates a complete-loss-of function mutation in the neurotransmitter-specific gene. In addition, the coding sequence for the LexA transcription factor is inserted such that after the B3 recombinase-mediated excision, a LexA driver is created that recapitulates the endogenous expression pattern of the neurotransmitter-specific gene. Although the B3 recombinase-mediated excision will occur in all neurons in which a given GAL4 driver expresses, the resulting LexA driver will express only in the subset of neurons in which a specific neurotransmitter is utilized. These neurons are easily visualized using a fluorescent reporter, but any transgene can be expressed using the LexA driver including transgenes for manipulating neuronal activity such as channelrhodopsin (for optogenetic excitation) or temperature-sensitive shibire (for neuronal silencing). In a genetic background otherwise mutant for a given neurotransmitter-specific gene, neurons within a GAL4 expression pattern that utilize that neurotransmitter will be silenced upon B3 recombinase-mediated excision. Definition of the neurotransmitter identity of each neuron within a neural circuit of interest, and an enhanced ability to manipulate the activity of small subsets of neurons based on the intersection of neurotransmitter usage and GAL4 expression pattern, will make possible finer scale neural circuit analysis than is currently possible with existing methods and thereby the potential for a more in- depth understanding of the functional properties of neural circuits.